The present invention generally relates to liquid chromatography, and specifically, to high-pressure liquid chromatography (HPLC) methods and systems for rapidly separating and/or characterizing a plurality of samples. The invention particularly relates, in a preferred embodiment, to hybrid parallel-serial HPLC methods and systems for separating and/or characterizing a combinatorial library comprising different polymers.
Liquid chromatography is generally well known in the art. High-pressure liquid chromatographic techniques involve injection of a sample into a mobile-phase that flows through a chromatographic column, separation of one or more components of the sample from other components thereof in the chromatographic column, and detection of the separated components with a flow-through detector. Approaches for liquid chromatography typically vary, however, with respect to the basis of separation and with respect to the basis of detection.
Gel permeation chromatography (GPC), a well-known form of size exclusion chromatography (SEC), is a frequently-employed chromatographic technique for separation of samples generally, and for polymer size determination in particular. Another chromatographic separation approach is illustrated by U.S. Pat. No. 5,334,310 to Frxc3xa9chet et al. and involves the use of a porous monolithic stationary-phase as a separation medium within the chromatographic column, combined with a mobile-phase composition gradient. Other separation approaches are also known in the art, including for example, normal-phase (e.g., adsorption) chromatography and reverse-phase chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, ion-exchange chromatography, affinity chromatography, among others.
After separation, a detector can measure a property of the sample or of a sample componentxe2x80x94from which one or more characterizing properties, such as molecular weight can be determined as a function of time. Specifically for polymers, for example, a number of molecular-weight related parameters can be determined, including for example: the weight-average molecular weight (MW), the number-average molecular weight (Mn), the molecular-weight distribution shape, and an index of the breadth of the molecular-weight distribution (MW/Mn), known as the polydispersity index (PDI). Other characterizing properties, such as concentration, size (e.g. for particles or polymers), architecture, chemical composition and/or chemical composition distribution can likewise be determined. A variety of continuous-flow detectors have been used for measurements in liquid chromatography systems. Common flow-through detectors include optical detectors such as a differential refractive index detector (RI), an ultraviolet-visible absorbance detector (UV-VIS), or an evaporative mass detector (EMD)xe2x80x94sometimes referred to as an evaporative light scattering detector (ELSD). Additional detection instruments, such as a static-light-scattering detector (SLS), a dynamic-light-scattering detector (DLS), and/or a capillary-viscometric detector (C/V) are likewise known for measurement of properties of interest.
Broadly available liquid chromatography systems are not entirely satisfactory for efficiently screening larger numbers of samples. With respect to polymers, for example, high-performance liquid chromatographic techniques can typically take up to an hour for each sample to ensure a high degree of separation over the wide range of possible molecular weights (i.e., hydrodynamic volumes) for a sample. Notably, however, substantial improvements in sample throughput have been achieved in the art. For example, rapid-serial approaches for characterizing polymers have been developed by Symyx Technologies, Inc. (Santa Clara, Calif.) and disclosed in the aforementioned co-pending U.S. patent applications from which the present application claims priority. As another example, U.S. Pat. No. 5,783,450 to Yoshida et al. discloses rapid-serial protocols and systems for preparation, purification and separation of small molecules such as catecholamines and protaglandins from biological samples such as blood.
Parallel approaches for liquid chromatography have also been contemplated in the art. Zeng et al., Development of a Fully Automated Parallel HPLC/Mass Spectrometry System for the Analytical Characterization and Preparative Purification of Combinatorial Libraries, Anal. Chem. 70, 4380-4388 (1998), disclose analytical and preparative HPLC methods and systems involving the sequential preloading of samples onto two chromatographic columns, and then applying a mobile-phase in parallel to each of the columns to effect parallel separation of the samples. According to an alternative approach disclosed in U.S. Pat. No. 5,766,481 to Zambias et al., parallel separation of a plurality of molecules is effected by forming a mixture of selected, compatible molecules, and subsequently resolving the mixture sample into its component molecules by separation in a single-channel HPLC system. Parallel approaches have likewise been employed in other separation protocols, such as capillary electrophoresis. See, for example, U.S. Pat. No. 5,900,934 to Gilby et al.
Although such parallel approaches and systems have been generally contemplated, there nonetheless exists a need in the art for improving such approaches and systems with respect to overall sample throughput and/or quality of data. Moreover, with the development of combinatorial materials science techniques that allow for the parallel synthesis of libraries comprising a vast number of diverse industrially relevant materials, and especially polymeric materials, there is a need for HPLC methods and systems to rapidly characterize the properties of samples from such combinatorial libraries.
It is therefore an object of the present invention to provide HPLC systems and protocols having a higher overall sample throughput, and in preferred applications, employing such systems and protocols for characterizing combinatorial libraries of material samples such as polymer samples, and particularly, libraries of or derived from reaction mixtures such as polymerization product mixtures, to facilitate the discovery of commercially important materials such as polymeric materials, catalysts, polymerization conditions and/or post-synthesis processing conditions.
Briefly, therefore, the present invention is directed to methods for separating and characterizing components of a plurality of samples with a high-performance liquid chromatography system. According to one preferred method, a mobile phase is supplied (e.g., pumped) in parallel through each of first and second chromatographic columns of a liquid chromatography system. First and second samples are serially injected into the mobile phase of the first and second chromatographic columns, respectively. At least one sample component of the injected first and second samples is then separated from other sample components thereof in the respective chromatographic columns. Preferably, in applications to analytical chromatography, a property of at least one of the separated sample components of the first and second samples is detected. A property of interest can then be determined from the detected property (e.g., by correlation to known standards for the property of interest).
The invention is also directed to several preferred variations of the aforedesribed method. In one preferred variation, four or more different samples are serially and distributively injected into a mobile phase being supplied in parallel to four or more chromatography columns. In another preferred variation of such method, ten or more different samples are serially loaded into an injection system (and preferably into an injector such as an injection valve), and then serially and distributively injected through a multi-port switching valve into one of the mobile phases being supplied in parallel to four or more chromatography columns. In each of the aforementioned methods, the number of parallel chromatographic channels is preferably at least four of more, and the number of samples (e.g., polymer samples) is preferably at least ten or more (and in some cases forty or more). In particular, for polymer screening, the invention is advantageously combined with rapid serial approaches applied in one or more of the parallel chromatographic channels.
The invention is directed as well to a liquid chromatography system useful for rapid separation and/or characterization of a plurality of samples. The system includes two or more chromatographic columns, and two or more supply conduits for providing parallel fluid communication between a liquid mobile-phase source and the two or more chromatographic columns, respectively. The liquid chromatography system also includes an injection system for serially and distributively injecting a plurality of samples into a liquid mobile phase supplied to the two or more chromatographic columns. The injection system comprises an injector and a multi-port switching valve. The injector has a sample-loading port (e.g., an injection port) for receiving a plurality of samples, and has a sample-discharge port for discharging the plurality of samples under pressure to the switching valve. The injector is preferably a multi-loop injection valve of the type known in the art. The switching valve has an inlet port and two or more selectable outlet ports. The inlet port of the switching valve is in fluid communication with the sample-discharge port of the injector, and is in selectable fluid communication with the two or more selectable outlet ports. The two or more selectable outlet ports are themselves in fluid communication with the two or more chromatographic columns, respectively, such that the injection system can serially and distributively inject the plurality of samples into the parallel-supplied mobile phase of the two or more chromatographic columns. A control system is preferably used to control the switching valvexe2x80x94that is, to control which of the two or more selectable outlet ports are in fluid communication with the inlet port. The system can also include one or more detectors having a flow cell in fluid communication with the chromatographic column effluentxe2x80x94for detecting a property of the plurality of samples or sample components. The system can also include an autosampler for loading the samples into the loading port/injection port of the injector.
In preferred embodiments, the system is a high-performance liquid chromatography system comprising four or more chromatographic columns, or eight or more chromatographic columns configured in parallel with respect to the mobile-phase flow through the columns. The system also preferably includes one or more microprocessors and one or more associated control systems or sub-systems for controlling the autosampler, injector, multi-port switching valve, and detectors, as well as the one or more pumps that supply the mobile-phase to the chromatographic columns.
Another aspect of the invention is directed to other applications of the aforementioned methods and systems for evaluating interactions between a plurality of liquid samples (e.g., samples dissolved in, dispersed in or emulsified in a liquid phase) and one or more solid materials or supported materials. Inverse chromatography with the single-injection/parallel mobile phase system is exemplary. More generally, however, the methods and systems of the invention can be applied to study solid/liquid interactions without regard to whether or not separation is effected.
The present invention provides substantial advantages over known approaches for parallel liquid chromatography systems. High overall throughput is achieved with a HPLC system involving time-based resolution of sample components without compromising data quality. In particular, the present invention overcomes limitations associated with the system disclosed by Zeng et al.xe2x80x94involving sequential pre-loading of the sample onto two columns, and subsequently initiating mobile-phase flow in parallel through the columns to effect parallel separation. Comparatively, the instant methods and system are more accurate and reproducible, since the methods and systems of Zeng et al.xe2x80x94inherently require an equilibration period once mobile-phase flow is initiated. Moreover, the instant methods and systems can be used with a broader range of detectorsxe2x80x94especially detectors that would be sensitive to or incompatible with the variation in mobile-phase flow (e.g., evaporative light scattering detectors (ELSD)), as well as with a broader range of rapid-serial techniques, such as overlaid injection, that require or advantageously employ continuous mobile-phase flow. Additionally, the prior art methods are inherently limited with respect to sample throughput. The time spent for stop-flow loading of samples onto or into the columns cannot, a priori, be used for separation, and as such, adversely affects the speed and overall throughput. Moreover, the interruption of flow cannot occur while the preceding sample is resident in the detector (e.g., in the flow cell) without adversely affecting the detector output for that sample. The systems of the instant invention are also comparatively more robust, since the chromatographic columns of the invention are not necessarily subjected to repeated mechanical stresses associated with stopping and initiating the mobile-phase flow.
Other features, objects and advantages of the present invention will be in part apparent to those skilled in art and in part pointed out hereinafter. All references cited in the instant specification are incorporated by reference for all purposes. Moreover, as the patent and non-patent literature relating to the subject matter disclosed and/or claimed herein is substantial, many relevant references are available to a skilled artisan that will provide further instruction with respect to such subject matter.